Susan M. Lea

Neisseria meningitidis recruits factor H using protein mimicry of host carbohydrates

The complement system is an essential component of the innate and acquired immune system, and consists of a series of proteolytic cascades that are initiated by the presence of microorganisms. In health, activation of complement is precisely controlled through membrane-bound and soluble plasma-regulatory proteins including complement factor H (fH; ref. 2), a 155 kDa protein composed of 20 domains (termed complement control protein repeats). Many pathogens have evolved the ability to avoid immune-killing by recruiting host complement regulators and several pathogens have adapted to avoid complement-mediated killing by sequestering fH to their surface. Here we present the structure of a complement regulator in complex with its pathogen surface-protein ligand. This reveals how the important human pathogen Neisseria meningitidis subverts immune responses by mimicking the host, using protein instead of charged-carbohydrate chemistry to recruit the host complement regulator, fH. The structure also indicates the molecular basis of the host-specificity of the interaction between fH and the meningococcus, and informs attempts to develop novel therapeutics and vaccines.

The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.

Heparan sulfate has an important role in cell entry by foot‐and‐mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre‐formed sulfate‐binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfate‐binding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.

The structure and antigenicity of a type C foot-and-mouth disease virus.

BACKGROUND Picornaviruses are responsible for a wide range of mammalian diseases and, in common with other RNA viruses, show considerable antigenic variation. Foot-and-mouth disease viruses (FMDVs) constitute one genus of the picornavirus family and are classified into seven serotypes, each of which shows considerable intratypic variation. This antigenic variation leads to continuing difficulties in controlling the disease. To date the structure of only one serotype, O, has been reported. RESULTS The three-dimensional structure of a serotype C (isolate C-S8c1) FMDV, has been determined crystallographically at 3.5 A resolution. The main chain conformation of the virion is very similar to that of type O1 virus. The immunodominant G-H loop of VP1, the presumed site of cell attachment, is disordered in both types of virus indicating a functional role for flexibility of this region. There are significant changes in the structure of other antigenic loops and in some internal regions involved in protomer-protomer contacts, including the entire amino-terminal portion of VP2, described here for the first time for a picornavirus. Antigenic sites have been identified by genetic and peptide mapping methods, and located on the capsid. The data reveal a major new discontinuous antigenic site (site D) which is located near to the three-fold axis and involves residues of VP1, VP2 and VP3 which lie adjacent to each other on the capsid. CONCLUSION In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans -activation and cis -inhibition

The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1DSL-EGF3). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1DSL-EGF3 binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

Structural basis for complement factor H–linked age-related macular degeneration

Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to ∼50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulators surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance–monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG–FH complex.

His-384 Allotypic Variant of Factor H Associated with Age-related Macular Degeneration Has Different Heparin Binding Properties from the Non-disease-associated Form

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.

Molecular model of a type III secretion system needle: Implications for host-cell sensing

Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are translocated, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex.

Dimerization of complement factor H-related proteins modulates complement activation in vivo

The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Self-Chaperoning of the Type III Secretion System Needle Tip Proteins Ipad and Bipd.

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.

What's the point of the type III secretion system needle?

Recent work by several groups has significantly expanded our knowledge of the structure, regulation of assembly, and function of components of the extracellular portion of the type III secretion system (T3SS) of Gram-negative bacteria. This perspective presents a structure-informed analysis of functional data and discusses three nonmutually exclusive models of how a key aspect of T3SS biology, the sensing of host cells, may be performed.